Anti-Obesity Product And Its Method Of Preparation

ABSTRACT

The present invention relates to compositions comprising one or more anthraquinones for use in the treatment of obesity and related metabolic and liver disease. More particularly it relates to a plant extract fraction rich in anthraquinones including both anthraquinone glycosides and anthraquinone aglycones obtainable from a member of the Leguminosae family, more particularly  Cassia  seeds. It also relates to a method of obtaining a plant extract fraction rich in anthraquinones.

TECHNICAL FIELD

The present invention relates to compositions comprising one or moreanthraquinones for use in the treatment of obesity, metabolic disease,or liver disease.

More particularly it relates to a plant extract fraction rich inanthraquinones including both anthraquinone glycosides (majoranthraquinone component, typically accounting for from 0.8-1.6% byweight of the starting material) and anthraquinone aglycones (minoranthraquinone component, accounting for up to 0.2% by weight of thestarting material) obtainable from a member of the Leguminosae family,more particularly Cassia seeds. It also relates to a method of obtaininga plant extract fraction rich in anthraquinones.

BACKGROUND ART

Obesity and their associated metabolic and liver diseases are a globalproblem. In the Western world, obesity has become a most pressing healthproblem, with over 20% of the population effected. In America 65% ofadults (127 million) are overweight or obese and healthcare costs forobesity and obesity-related conditions are estimated to amount to $100billion.

The problem is growing in Europe. In the UK about 43% of men and 34% ofwomen are overweight with a further 22% of men and 23% of women beingclinically obese. There is a rising level of premature obesity inchildren and obesity is increasing in the developing world.

Anti-obesity medicines are usually divided into categories by mechanismsof action, namely those which:

-   -   (1) Reduce the feeling of hunger or suppress appetite through        the central brain;    -   (2) Inhibit fat absorption through affecting the digestive        system;    -   (3) Increase the energy consumption through capillary ending        mechanism; and    -   (4) Stimulate the metabolic function of the capillary ending of        adipose tissues to decrease the fat quantity or reduce synthesis        of triglyceride.

Sibutramine and Orlistat are two anti-obesity drugs approved by theAmerica FDA. These two drugs have the largest market shares inweight-loss medicines in the world.

The applicant has sought a natural solution to the problem of obesity.

Although Yang et al (Chinese Journal of Clinical Rehabilitation, 21 Aug.2005 Vol. 9, No. 31) observed that a decoction (water extract) ofCassia-seed inhibited the weight gain, in rats with nutritional obesity,the active compounds or fraction of Cassia seeds which related to theactivity of inhibiting the weight gain has not previously beenidentified.

Other prior art includes:

J. Natural products, 1990, Vol. 53, No. 3, pp. 630-633 which discloses ahexane extraction followed by a methanolic fractionation of Cassia seedsto obtain sub-fractions which showed activity as inhibitors of plateletaggregation. The three active fractions contained respectivelygluco-obtusifolin, gluco aurantio-obtusifolin and gluco-chrysobtusin.

CN1733679 which discloses a method of extracting anthroquinone analogcompounds from Cassia seeds. The compounds extracted areanti-hyperglycemic ingredients.

JP0214915 discloses aldose reductase inhibitors containing anthraquinonecompounds used in treating complications in diabetes.

Arch. Pharm. Res. Vol. 19, No. 4, p 302-306, 1996 discloses NMRassignments of anthraquinones isolated from Cassia tora.

J Chinese Medicinal Materials, 2004, vol. 24 pp. 758-764 discloses awater extract of Cassiae seeds. It reports that obese rats showed weightloss, and metabolism of glucose and lipid.

International Journal of Obesity, 2000, vol. 24, pp. 758-764 disclosesan extract CT-II of Cassia mimosoides L var nomame showed an inhibitoryeffect on lipase. The extract was a fraction of an aqueous ethanolicextract.

J. Traditional Chinese Med, 1995, vol. 15, no. 3 pp. 178/9 discloses atrial of a 5 herb mix including Cassia Tora in the treatment ofhyperlipemia. The mix was shown to reduce serum cholesterol andtriglycerides as well as bodyweight.

Progress in nutrition, 4, 2, 147-150, 2002 reports on a trial ofChitosano and Cassia Noname var Mimosoides on obese patients.

BE 1009545 discloses food supplements for slimming purposes comprising acombination of at least three vegetable extracts from a selection ofseven, one of which is senna pods (Cassia sp).

The applicant has identified Chinese Cassia seeds as a promisingcandidate and has, for the first time, carried out systematic studies onCassia seeds in accordance with the regulatory requirements of theChinese Food and Drug Administration (CFDA) in the following areas:

1. Botany and Pharmacognosy

2. Phytochemistry

3. Pharmacology and Toxicology

4. Pharmaceutical processes

5. Analytical chemistry

6. Pharmaceutics

The botanical terms used herein are intended to have the meanings asused by the FDA in their directive “Guidance for Industry—Botanical DrugProducts” (June 2004) which directive is incorporated by reference. Itwill, however, by understood by persons skilled in the art that indifferent countries different terminology may be used. Reference to theFDA guidance terminology is used for consistency and should not be takento be limiting. Thus, differing but equivalent terms used by, forexample, the CFDA or EMEA in their Guidelines on Quality of HerbalMedicinal Products/Traditional Herbal Medicinal Products (CHMP/THMPadopted March 2006), will be understood by the skilled person to beencompassed by the terms used herein.

DISCLOSURE OF THE INVENTION

According to a first aspect of the present invention there is providedplant extract, from a member of the Leguminosae family, comprising byanalysis:

i) at least 1% aurantio-obtusin (by hplc analysis);

ii) at least 0.05% obtusifolin (by hplc analysis); and

iii) at least 10% total anthraquinone (by UV spectroscopy).

Preferably the plant from the Leguminosae family is from a Cassia spp.

Preferably the Cassia spp. is selected from:

1. Cassia obtusifolia, or

2. Cassia tora

although other Cassia species including Cassia occidentatis L might beused. Most preferred is Cassia obtusifolia.

Preferably the plant material used is the seed.

The preferred plant extract can be defined by reference to markers. Thepreferred markers are aurantio-obtusin and obtusifolin.

Preferably, aurantio-obtusin is present in an amount (by HPLC analysis)that constitutes at least 1% of the extract. More preferably the contentof aurantio-obtusin, measured as and calculated on the basis of thetotal aglycone, after acid hydrolysis, constitutes at least 2%,increasing in integers of 1%, through 3%, 4% to most preferably at least5% and may increase further through 6%, 7%, 8%, and 9% to as much as 10%or higher.

Preferably, obtusifolin is present in an amount (by HPLC analysis) thatconstitutes at least 0.1%, of the extract. More preferably the contentof obtusifolin, measured as and calculated on the basis of the totalaglycone after acid hydrolysis, constitutes at least 0.2%, increasing inintegers of 0.1%, through 0.3%, 0.4%, to 0.5% and may increase furtherthough 0.6% and 0.7% to as much as 0.8% or higher.

This can be determined using HPLC and/or methodology as described in thedetailed description.

The preferred composition comprises a defined ratio mix of the markers

1. Aurantio-obtusin; and

2. Obtusifolin.

Most preferably the two markers are present in a ratio ofaurantio-obtusin:obtusifolin of between 5:1 to 30:1, more preferably 5:1to 16:1 and most preferably between 8:1 and 16:1. Most preferred is aratio of greater than 8:1.

Such a ratio of markers may differ from that naturally found in theplant as can be seen from Example 1 which discloses a method forobtaining and purifying the respective markers from an ethanolicextract. It will be noted that the aurantio-obtusin is obtained fromfraction A at levels of eight times that of the obtusifolin found infraction B. In contrast, the present invention preferably uses afraction with higher relative proportions of aurantio-obtusin toobtusifolin. (Preferably greater than 8:1, and most preferably between8:1 and 16:1).

Preferably the extract comprises a total anthraquinone content of atleast 25%, more preferably at least 30%, through 35%, 40%, 45% to 50% ormore (by UV spectrometry).

The anthraquinones associated with Cassia spp are illustrated in thetables 1 and 2 below:

TABLE 1 Table 1 Chemical components of anthraquinones from Cassiaobtusifolia L. and Cassia tora L. Cassia obtusifolia Cassia tora Emodin1 1 Chrysophanol 2 2 Rhein 3 3 Physcion 4 4 Ale-emodin 5 5 Chrysarobin 66 Chrysophanic acid anthrone 7 7 Obtusifolin 8 10 Obtusin 9 11Chryso-obtusin 10 Chrysophanol-1-β-geniobioside 28 Aurantio-obtusin 111-[(β-D-glucopyranosyl-(1→ 3)-O- 29 Questin 12 β-D- Gluco-obtusifolin 13glucopyranosyl(1→ 6) O-β-D- glucopyranosyl)oxy]-8-hydroxy-3methyl-9,10-anthraquinone Gluco-obtusin 14 1-[(β-D-glucopyranosyl-(1→6)-O- 30 Gluco-aurantioobtusin 15 β-D- 1-Demethyl auratioobtusin 16glucopyranosyl(1→ 3) O-β-D- glucopyranosyl(1→ 6)-O-β-D-glucopyranosyl)oxy]-8-hydroxy-3 methyl-9,10-anthraquinone1-Denethylobtusin 17 2-(β-D-glucopyranosyloxy-8- 31 hydroxy- 1-methoxy-3methyl-9,10- anthraquinone 1-Demethyl chryso-obtusin 18Alaterin-2-O-β-D-glucopyranoside 32 Chrysophanol 10, 10-bianthrone 19Echinul polydric anthrone-8-O-D- 20 glucopyranosideAlaternin-1-O-β-D-glucopyranoside 21 Emodin-6-glucoside 22 Emodinanthrone 23 Chryso obtusin-2-O-β-D-glucopyranoside 24 Chryscion8-O-D-glucopyranoside 25 1,3-dihydroxy-6-methoxy-7-methyl- 26anthraquinone 1-hydroxy-3,7-diformyl-anthraquinone 27

TABLE 2 List of chemical structural formula of partial anthraquinonesfrom Cassia Seeds

No. R₁ R₂ R₃ R₆ R₇ R₈ 1 OH H CH₃ OH H OH 2 OH H CH₃ H H OH 3 OH H COOH HH OH 4 OH H CH₃ OCH₃ H OH 5 OH H CH₂OH H H OH 8 OCH₃ OH CH₃ H H OH 9OCH₃ OH CH₃ OCH₃ OCH₃ OH 10 OCH₃ OH CH₃ OCH₃ OCH₃ OCH₃ 11 OCH₃ OH CH₃ OHOCH₃ OH 12 OH H CH₃ OH H OCH₃ 16 OH OH CH₃ OH OCH₃ OH 17 OH OH CH₃ OCH₃OCH₃ OH 18 OH OH CH₃ OCH₃ OCH₃ OCH₃ 21 O-glu OH CH₃ OH H OH 22 OH H CH₃O-glu H OH 24 OCH₃ O-glu CH₃ OCH₃ OCH₃ OCH₃ 25 OH H CH₃ OCH₃ H O-glu 26OH H OH OCH₃ CH₃ H 27 OH H CHO H CHO H 28 O-gen H CH₃ H H OH 29O-{circle around (1)} H CH₃ H H OH 30 O-{circle around (2)} H CH₃ H H OH31 OCH₃ O-glu CH₃ H H OH 32 OH O-glu CH₃ OH H OH

Preferably the extract is an extract which has been purified such thatit has been purified by at least a factor of 10, compared to the dryweight of the starting materials, and more preferably at least a factorof 25, through 50, 75 and 100. Thus, preferably it contains less than10% more preferably still less than 4% through 2% and most preferablyless than 1% by weight of the starting material.

The extract obtained is primarily designed as a Botanical Drug Substance(BDS) with the intended use as an active pharmaceutical ingredient (API)to be made into a pharmaceutical dosage form or Botanical Drug.Alternatively the extract may be used as an ingredient of a dietarysupplement or health foodstuff where its inclusion may be to bring abouta therapeutic or cosmetic effect.

In order to obtain a characterized extract according to the inventionthe Applicant has had to overcome a number of problems including:

-   -   1. Ensuring quality assurance, QA, and quality control, QC, of        the starting raw material;    -   2. Developing novel extraction and purification processes;    -   3. Defining the specification of the extract;    -   4. Developing appropriate analytical methodology specific to        both the raw material and the extract;    -   5. Identifying relevant chemical markers;    -   6. Producing standard reference chemicals;    -   7. Identifying the active fraction and it's characteristics;    -   8. Determining the activity so as to produce an appropriate        final dosage form; and    -   9. Determining the toxicity of the extract.

When the extract is used as a BDS, it is preferably formulated for oraldelivery. It may be taken in such forms as a powder, solution,suspension, tablet, lozenge, granule or capsule. A capsule dosage formcontaining a dried extract has been shown to exhibit therapeuticactivity.

The extract preferably takes the form of a dried extract powder, whichmay be taken as a daily dose in an amount of from 50-5,000 mg. A typicaladult dose will be 500 mg (equivalent to a daily intake of 50 g of rawmaterial).

Preferably the plant species is a Cassia spp selected from:

1. Cassia obtusifolia; or

2. Cassia tora.

Most preferably the plant species is Cassia obtusifolia and moreparticularly still Cassia obtusifolia grown in the habitat of HubeiProvence of China since plant material from this region has been foundby the applicant to contain a higher percentage of aurantio-obtusin,measured as and calculated on the basis of the total aglycone after acidhydrolysis, than plant material from other regions (greater than 0.06%,typically around 0.08%). Alternatively, the seeds of C. obtusifoliagrown in Shanxi and Anhui Provinces may also be used.

The preferred plant material is seed.

Preferably the fraction is obtained by way of an ethanolic extraction,most preferably a reflux extraction as such a method gives significantlyhigher concentrations of the marker compounds and total anthraquinonesthan alternative methods.

Preferably the method includes steps to remove:

-   -   i) Any lipo-soluble oils; and    -   ii) Water soluble gums.

This may comprise respectively:

-   -   i) A separation with super-speed centrifugation or partitioning        with lipo-soluble solvents, such as, for example petroleum        ether, chloroform, etc; and    -   ii) Washing in water and/or a low concentration of ethanol.        (Less than 30% by volume).

Preferably a resin absorption separation and purification process isconducted using a macroporus resin column. The favored macroporous resinis a non-polar styrol copolymer resin, such as, for example (D101)produced by Tianjin Agricultural Chemical Corp. Ltd, Resin Branch. Thishas the following specifications:

Product Name Macroporous Resin

Chemical Name Styrol Copolymer

Brand (Type): Yuanhang Brand (D101)

Structure: PSD

Appearance: Milky white or light yellow opaque global granule

Polarity: Non-polarity

Specific surface area: 400 m²/g

Grain size: 20-60 mesh≧90%

Moisture: 65-75%

Average pore diameter: 0.0301 μm

Porosity: 70.5%

Pore volume: 1.6824 ml/g

Wet true density: 1.1778 g/cm³

Apparent density: 0.65-0.70 g/ml

Dry density: 0.3475 g/cm³

Skeleton density: 0.8369 g/cm³

Other advantageous process conditions will be apparent from thesupporting data given in the detailed description. These include:

-   -   1. The use of a 50-80% ethanolic solution for extraction;    -   2. The optimization of reflux extraction conditions;    -   3. The pre-treatment process for column purification;    -   4. The selection of a preferred macroporous resin together with        selected column dynamics: e.g. height ratio of the resin bed        should be 1:5-1:50;    -   5. The control of elution conditions to remove impurities e.g.        volume/concentration/speed e.g. Elute with 2 BV (bed volume) of        distilled water, and then with 1-8 BV of 20% ethanol at a speed        of 1-5 BV/h; and    -   6. The control of elution conditions to control anthroquinone        separation e.g. Elute with 1-10 BV of 70% ethanol at the speed        1-5 BV/h, collect the ethanol eluent.

According to further aspects of the invention there are provided aBotanical Product (including a food, drug or cosmetic), such as, forexample a Botanical Drug Product, Botanical Drug, Botanical drugsubstance, Cosmetic, or Dietary supplement. The Botanical Drugpreferably takes the form of an oral dosage form, preferably deliveredas a dry powdered extract in a capsule.

According to yet further aspects of the invention there are provide theuse of a plant extract, from a Leguminosae family, in the manufacture ofa medicament for the treatment of obesity, metabolic disease or liverdisease; the use of a plant extract, from a Leguminosae family, in themanufacture of a cosmetic preparation for the treatment of obesity andthe use of a plant extract, from a Leguminosae family, in themanufacture of a functional food or dietary supplement.

According to still yet further aspects of the invention there areprovided a method for the treatment of obesity, metabolic disease orliver disease comprising administering a plant extract, from aLeguminosae family; a method for the cosmetic treatment of obesitycomprising administering a plant extract, from a Leguminosae family; anda method for supporting weight loss comprising administering afunctional food or dietary supplement comprising a plant extract, from aLeguminosae family.

The various aspect of the invention will be further described, by way ofexample only, with reference to the following Figures and Examples:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram showing a method of obtaining pure samples ofthe markers aurantio-obtusin and obtusifolin from Cassia seed;

FIG. 2 is an HPLC chromatogram for the markers;

FIG. 3 is a flow diagram showing an extraction method according to oneaspect of the invention;

FIG. 4 is an HPLC chromatogram for an extract of the invention;

FIG. 5 is a TLC plate of an extract of the invention at 365 nm;

FIG. 6 is a TLC plate of an extract of the invention at 254 nm;

FIG. 7A is a graph showing the effect of PYN22 on weight;

FIG. 7B is a graph showing the effect of PYN22 on weight gain;

FIG. 7C is a graph showing the effect of PYN22 on body fat;

FIG. 5A is a graph showing the effect of PYN22 on bloodcholesterol—total cholesterol;

FIG. 8B is a graph showing the effect of PYN22 on bloodcholesterol—HDL-cholesterol;

FIG. 9A is a graph showing Fasting insulin levels at day 7;

FIG. 9B is a graph showing Fasting insulin levels at day 21;

FIG. 9C is a graph showing Fasting insulin levels at day 35;

FIG. 10A is a graph showing Oral Glucose Tolerance Tests (AUC) at day 7;

FIG. 10B is a graph showing Oral Glucose Tolerance Tests (AUC) at day21;

FIG. 10C is a graph showing Oral Glucose Tolerance Tests (AUC) at day35;

FIG. 11A is a graph showing the effects of PYN22 on liver indicators atday 43—inflammatory markers—ALT;

FIG. 11B is a graph showing the effects of PYN22 on liver indicators atday 43—inflammatory markers—AST;

FIG. 11C is a graph showing the effects of PYN22 on liver indicators atday 43—inflammatory markers—liver weight;

FIG. 11D is a graph showing the effects of PYN22 on liver indicators atday 43—inflammatory markers—as percent body weight;

FIG. 11E is a graph showing the effects of PYN22 on liver indicators atday 43—inflammatory markers—liver glycogen; and

FIG. 11F is a graph showing the effects of PYN22 on liver indicators atday 43—inflammatory markers—tri-glyceride content.

DETAILED DESCRIPTION 1.0 Starting Materials 1.1 Background

Cassia seeds (both Cassia tora L. and Cassia obtusifolia L.) containanthraquinones, naphtha-pyrones, fatty acids, amino acids and inorganicelements. The main constituents are anthraquinones including 0.8-1.6%anthraquinone glycosides and 0.06-0.2% anthraquinone aglycones.

Cassia seeds usually refer to mature dried seeds of Cassia tora L. andCassia obtusifolia and are widely available. Cassia occidentalis on theother hand is only used in some local areas of southern China.

According to the distribution of the plants, Cassia tora L. isdistributed in the provinces south of the Yangtze River, and can not becultivated in the northern area as it is not able to bear flowers orfruits. On the other hand Cassia obtusifolia L. can grow both in thenorth and south. The literature indicates that in ancient time, Cassiaseeds were from the above three species.

Research on the chemical components of Cassia, has primarily beencarried out by Japanese scientists with Japanese Cassia seeds. Some ofthe prominent chemicals isolated from Japanese Cassia, e.g. rhein andaloe-emodin, don't seem to exist in Chinese Cassia seeds. Scientistshave so far paid very little attention to the chemical profile ofChinese Cassia seeds, except the knowledge of the existence of a fewfrequently occurring Rheum anthraquinones. The active component of, andchemicals specific to Chinese Cassia seed, are as yet to be identified.At the moment the quality control method for raw material of Cassiaseeds and its preparation is based on the determination of the contentof Chrysophanol and total anthraquinones. The former ubiquitously existsin many medicinal plants containing anthraquinone derivatives while themeasurement of the latter is even less specific.

Through a systematic phytochemical study on Cassia seeds, the applicanthas determined that plant material from North China mainly containedchrysophanol, physcion, obtusifolin, emodin, and aruantio-obtusin. Twoof them, aruantio-obtusin and obtusifolin seem to be the characteristicchemicals of Chinese Cassia seeds, while aruantio-obtusin was found tobe the most prominent chemical in those fractions which produced amarked weight loss effect.

1.2 Selection of the Raw Materials

Based on the applicants' research, the amounts (by weight) of the activeconstituents in Cassia tora were found to be lower than those in Cassiaobtusifolia. So Cassia obtusifolia was selected for furtherinvestigation.

There are many places producing Cassia species and the contents of theanthraquinones and aurantio-obtusin varied significantly. The applicantinvestigated the samples from Guangxi, Anhui and Hubei provinces inorder to identify a most appropriate production site and to ensure theconsistent quality of the raw materials.

The raw materials from the above locations were quantified for theircontent of the anthraquinones and aurantio-obtusin, measured as, andcalculated on the basis of, the total aglycone after acid hydrolysis, byUV spectrophotometry and HPLC methods, respectively.

The results of the investigation are illustrated in Table 3 below:

TABLE 3 Table 3 Quantitative assay for raw materials from differentlocations Raw material Anthraquinones Aurantio-obtusin Location Mean %Mean % Guangxi 0.8 0.051 Anhui 1.1 0.071 Hubei 1.4 0.080

From table 3 it will be noted that the chemical content of the rawmaterials varied with location. Based on the results applicant selectedthe Cassia obtusifolia raw materials from Hubei province.

2.0 Compounds

The marker compounds used to characterize the plant extract of theinvention are noted below:

Registry Number: 67979-25-3

and

Registry Number: 477-85-0 3.0 Extraction

3.1 Method of Separation and Purification of Obtusifolin andAurantio-Obtusin from Cassia Seeds

Referring to FIG. 1, 1 Kg of cassia seeds were extracted with ethanol.The solution was filtered and the ethanol recovered under reducedpressure leaving a residue and extract (190 g).

The extract was partitioned with chloroform producing a chloroformlayer—Fraction A and the mother solution—Fraction B.

Fraction A (20 g) was absorbed onto a silica gel column, eluted withchloroform-methanol in gradient. The corresponding eluent was collectedand re-crystallized with methanol. It contained 0.1 g of obtusifolin.

Fraction B was absorbed onto a macroporous resin column and eluted withethanol. The eluent was further separated on a silica gel column andeluted with chloroform-methanol in gradient. The corresponding eluentwas collected and re-crystallized with methanol. It contained 0.5 g ofaurantio-obtusin.

3.1.1 Quantitative Assay 3.1.2 Equipments and Materials

HPLC Agilent 1100 Series, diode-array multi wavelength detector.

Methanol and acetonitrile (chromatographic grade), dc-ionized water andphosphoric acid (analytical grade).

3.1.3 Method and Result

Chromatography Conditions:

Mobile Phase:

Eluted with methanol-water, methanol-0.1% phosphate, acetonitrile-water,and acetonitrile-0.1% phosphate in gradient, respectively, at differentflow rates as shown in Table 4.

TABLE 4 Table 4 The gradient of the mobile phase Time ACN (%) 0.1% H₃PO₄FLOW (ml/min) 0 40 60 0.8 5 50 50 0.8 20 100 0 0.8

Detection wavelength: with DAD detector, reference standards have theabsorption at 278 nm with the reference wavelength at 360 nm.

Chromatographic column: lunar C₁₈

Temperature: 25° C.

Running time: 25 mins.

3.1.4 Preparation of the Test Solutions:

Aurantio-obtusin and Obtusifolin reference standards were weighedaccurately and put into 10 ml volumetric flasks, respectively. Methanolwas added to dissolve the chemicals thoroughly to volume.

Retention time of Obtusifolin was determined to be 14.512 minutes andAurantio-obtusin determined to be 11.479 minutes (FIG. 2).

TABLE 5 Table 5 Linear relationship between the HPLC integration peakareas and the reference sample concentrations Correlation CompoundsLinear Relationship Coefficient Range (μg) Aurantio- Y = 93.3885334x-0.99999 0.092~1.580 obtusin 15.328363 Obtusifolin Y = 123.192564x-0.99995 0.008~0.580 3.4891702

3.2 Preparation of a Plant Extract (Best Mode)

Referring to FIG. 3 there is illustrated the preferred embodiment of amulti-stage process for preparing a plant extract of the invention. Itcomprises the following steps:

-   -   3.2.1 Pulverize Cassia seeds (25 Kg) into a coarse powder;    -   3.2.2 Carry out a reflux extraction with 4-20 folds in volume of        50-80% ethanol for 0.5-3 hours;    -   3.2.3 Repeat 2-4 times;    -   3.2.4 Filter and recover ethanol from the solution under vacuum        to obtain a crude extract;    -   3.2.5 Centrifuge the residue to remove any lipo-soluable oils;    -   3.2.6 Adjust concentration of the extract with distilled water        to the ratio 1:1-1:10 (material: solution);    -   3.2.7 Carry out separation and purification with a column filled        with D₁₀₁ Macroporous Resin at the ratio 1:2-20 (diameter:        height);    -   3.2.8 Apply the extract with a quantity of less than 1-4 BV (BV:        resin bed volume) to the column with dynamic absorption at the        speed of 1-2 BV/h;    -   3.2.9 Wash with 1-8 BV of water and then with 1-8 BV of 20%        ethanol at a speed of 1-5 BV/h;    -   3.2.10 Elute with 1-10 BV of 70% ethanol at the speed of 1-5        BV/h and collect the ethanol solution;    -   3.2.11 Recover ethanol and dry the extract under vacuum;    -   3.2.12 Dissolve the extract in 95% ethanol. Filter and recover        the ethanol from the solution to obtain a concentrated extract;    -   3.2.13 Dry the refined extract under vacuum and then pulverize        to give particles of a desired size for filling into capsules        (size 1).

The resulting extract is a dark brown powder with a bitter taste. It issoluble in water, ethanol and methanol.

The preferred extraction conditions were selected based on the followingfindings:

3.3. Selection of Ethanol Concentration

Pilot scale extractions were run at ethanol concentrations ranging from0% to 80%, in 10% increments, to determine optimum conditions.

Method: Weigh 50 g of Cassia seed coarse powder and add 300 ml ofethanol at selected concentrations, respectively. Carry out reflexextraction for 1.5 hours, and repeat 3 times. Filter and adjust thefiltrate to 1000 ml.

Measurements: The content of aurantio-obtusin, and the anthroquinonefraction were quantified and yield measures obtained.

The results are illustrated in Table 6 below:

TABLE 6 Table 6. The effect of ethanol concentration on the extractionof Cassia seeds Aurantio- Total Ethanol obtusin anthraquinone Yield conc(mean) (mean) (mean) 0 0.16 5.62 8.61 10 0.18 5.56 8.71 20 0.17 5.587.91 30 0.19 6.95 8.58 50 0.20 8.94 8.54 60 0.21 9.48 8.81 70 0.21 9.208.25 80 0.22 9.95 8.49

From the above it can be seen that the percentage auratonin-obtusin andtotal anthraquinone content increased with increasing ethanolconcentration but extract yield decreased. The optimum combination wasat around 60%.

3.4 Selection of Ethanol Extraction Method

Three commonly used ethanol extraction methods: percolation,cold-maceration and reflex extraction, were compared:

Percolation method: Weigh 50 g of Cassia seed coarse powder and add 100ml of 60% ethanol for 12 hours. Percolate with 500 ml ethanol andcollect the solution, adjust the volume with ethanol to 1000 ml.

Cold-maceration: Weigh 50 g of Cassia seed coarse powder and add 100 mlof 60% ethanol to macerate for 12 hours. Filter the solution undervacuum. Add ethanol 250 ml to the residue and macerate for another 12hours. Filter the solution under vacuum. Combine the two filtrates.Adjust the solution with the solvent to 1000 ml.

Reflux extraction: Weigh 50 g of Cassia seed coarse powder and add 300ml of 60% ethanol and carry out reflux extraction for 1.5 hours. Add 250ml ethanol and carry out the extraction for another 1.0 hour. Combinethe two filtrates and adjust the solution with the solvent to the volume(1000 ml).

The results are as illustrated in Table 7 below:

TABLE 7 Table 7 Effect of different methods on the extraction of Cassiaseeds Extraction method Aurantio-obtusin Anthraquinone Yield Percolation0.38 2.84 8.37 Maceration 0.23 1.48 5.12 Reflux 0.86 5.92 23.47

Reflux extraction resulted in significantly higher concentrations of themarkers and yields than the other methods.

3.5 Volume/Time/Frequency Analysis (ABC)

An orthogonal test was devised to determine the optimumVolume/Time/Frequency for the extraction. The results (not shown)indicated that only frequency had a significant effect and accordinglyreflux was repeated. The selected criteria were to use 8 folds ofethanol, to reflux for 1 hour and to repeat the process 3 times.

3.6 Purification

The optimum purification conditions after the oils were removed withcentrifugation were determined to be as follows:

-   -   3.6.1 Conduct separation and purification with column filled        with D₁₀₁ Macroporous Resin;    -   3.6.2 Employ a column ratio (diameter to height) of 1:20;    -   3.6.3 Add the sample to the column with a dynamic absorption of        less than 1.4 BV (BV: resin bed volume) preferably at a speed of        1 BV/h.    -   3.6.4 Wash with 2 BV of water and then with 4 BV of 20% ethanol        at a speed of 2 BV/h (This step is used to remove water soluble        gums);    -   3.6.5 Elute with 4 BV of 70% ethanol at a speed of 2 BV/h and        collect 70% ethanol solution;    -   3.6.6 Recover ethanol and dry the residue under vacuum;    -   3.6.7 Dissolve the extract in 95% ethanol. Filter and recover        the ethanol from the solution under vacuum;    -   3.6.8 Dry the residue at 60° C. and pulverize to a desired        particle size.

Fuller details and evidence supporting the selection of the aboveconditions are given below:

3.7 Selection of Resin

Both static and dynamic absorption ability was investigated in severalresins. The selection criteria were the absorption and desorptionefficiency of the resins.

3.7.1 Pre-Treatment of Resin

Clean the column to prevent contamination. Add half of the resin volumeof 95% ethanol into the column first, and then fill the column withmacroporous resin. The level of ethanol solution should be 0.3 meterabove the resin bed top. Leave the ethanol solution in the column for 24hours.

Use 2 BV (2 folds of Bed Volume of resin in the column, same as below)of 95% ethanol to wash the resin at a speed of 2 BV/h. Leave thesolution in the column for a further 4-5 hours. Wash the resin againwith 95% ethanol at a speed of 2 BV/h until the eluent shows nocloudiness when diluted with 5 times of water. Then wash the resin withdistilled water at the same speed until the eluent does not smell ofethanol.

Use 2 BV of 5% HCl solution to wash the resin at a speed of 4-6 BV/h andleave the solution in the column for 2-4 hours. Use distilled water towash the resin again at the same speed until the eluent has a neutral pHvalue.

Use 2 BV of 2% NaOH solution to wash the resin at the speed of 4-6 BV/hand leave the solution in the column for 2-4 hours. Use distilled waterto wash the resin again at the same speed until the eluent has a neutralpH value.

3.7.2 Static Absorption Test

Method: The five resins selected were treated to remove surface water.

A quantity of each resin was weighed and put into a stopped flask. Thetest solution was added to a hyper-saturated status and vibrated for 24hrs to enable the resin to fully absorb the anthraquinones. Quantify thecontent of the anthraquinones to evaluate the efficiency of the staticabsorption ability of the resin.

Filter and obtain filtrate 1. Add 80 ml of 95% ethanol into the resinand vibrate for 24 hrs to desorb the compounds, then filter again andobtain the filtrate 2.

Determine the contents of filtrate 1 and 2 (not given), and calculatethe absorption and desorbtion rates. The result are given in Table 8below

TABLE 8 Table 8. The absorbing and desorbing rate of anthraquinones onmacroporous resin in the static absorbing test Chemical Absorbing rateDesorbing rate Resins contents (%) (%) tested (mg) mean mean D₁₀₁ 126035.72 94.81 X-5 1260 27.78 88.81 NKA-9 1260 19.05 65.45 NKA-12 126021.95 95.56 D4020 1260 21.51 61.14

It can be seen from Table 8 that the best all round macroporus resin wasD₁₀₁ as it was bound relatively efficiently (greater than 30%) and inexcess of 90% of the bound material was desorbed.

3.7.3 Dynamic Absorption Test

Method: The eluent from the column was subjected to HPLC. The additionof test solution to the column was stopped when aurantio-obtusin wasdetected. Record the volume of the test solution added. Wash the columnwith distilled water until the eluent is nearly colorless and collectthe water solution. Then elute with 95% ethanol until the eluent isnearly colorless and collect the ethanol solution. Determine the contentof the anthraquinones in these two solutions and calculate theabsorption and desorbtion rate, respectively. The results are providedin table 9

TABLE 9 Table 9. The absorbing and desorbing rate of anthraquinones onmacroporous resin in the dynamic absorbing test Resins AnthraquinonesAbsorbing rate (%) Desorping rate (%) tested (mg) mean mean D₁₀₁ 336064.28 85.12 X-5 3360 56.38 96.87 NKA-9 3360 27.18 54.76 NKA-12 336058.52 90.69 D4020 3360 38.09 58.93

In the dynamic absorption test D101 resin showed better ability in bothabsorption and desorbtion than the others. In this regard the absorbingrate was in excess of 60% and the desorbing rate in excess of 80%.

The static and dynamic absorption test results suggested that D₁₀₁macroporous resin was better than the others so it was selected forseparation and purification of the crude extract.

3.8 Investigation of Technical Parameters in the Column Separation andPurification of the Anthraquinones

Concentration of the sample application to the column.

Sample solutions with different concentrations (raw material: solution)were flowed through the column filled with 50 g of pre-treated D₁₀₁resin as for the dynamic absorption test. The absorbed quantity of theanthraquinones was determined. The results are given in Table 10 below.

TABLE 10 Table 10. Absorbed quantities of anthraquinones on D₁₀₁macroporous resin in different concentration of the Cassia extractsDesorping rate (%) Sample concentration mean 1:1 47.22 1:2 51.75 1:576.66 1:8 76.61  1:10 76.61

From the above table it can be seen that the anthraquinones were moreeasily absorbed to resin when the concentration ratio increased andplateaued at 1:5 (raw material:solution).

3.9.1 Determination of Absorption Flow Speed

The sample solutions were flowed through the column filled with 50 g ofpre-treated D₁₀₁ resin at the speeds of 1, 2, 3 BV/ht, respectively, fordynamic absorption test. Elute with distilled water and then 95%ethanol, collect the ethanol solution to determine the content of theanthraquinones.

The results are given in Table 11 below:

TABLE 11 Table 11. The effect of flow speed on the absorption of D101macroporous resin for Cassia extracts Speeds 1 BV/h 2 BV/h 3 BV/h Mean2140 1970 1773 anthraquinone content mg

The results suggested that the slower the speed, the better theabsorption effect. Thus the flow speed of 1 BV/h was selected.

3.9.2 Leakage Curve Determination

The sample solution was flowed through a column filled with 50 g ofpre-treated D₁₀₁ resin with the conditions as for the dynamic absorptiontest. The eluent was collected in 10 ml fractions, and 10 fractions intotal were collected. Filter the fractions with 0.2 μm microporous filmand determine the content of aurantio-obtusin by HPLC. The result aregiven in table 12.

TABLE 12 Table 12. Leakage curve of Cassia extracts on D101 macroporousresin. Fractions ml 50 10 20 30 40 (1BV) 60 70 80 90 100 aruantio- 0 0 00 0 0 0 45.00 96.75 97.75 obtusin mg

From the table above, the leakage started when the sample quantity inthe column exceeded 1.4 BV. (ml/g)

3.10 Investigation of the Concentration of the Eluent

The sample solutions were flowed through 4 columns filled with 50 g eachof pre-treated D₁₀₁ resin with conditions as for the dynamic absorptiontest. The 4 columns were eluted with distilled water until the eluentwas nearly colorless and then eluted with 30%, 50%, 70% and 90% ethanol.The ethanol eluent was collected. The content of the anthraquinones wasdetermined. The result are given in table 13.

TABLE 13 Table 13. Effect of different concentration of ethanol on theelution of anthraquinones Concentration 30% 50% 70% 90% Anthraquinones1587 1907 2140 1733 mg

The result showed that the fraction between 50 and 90% ethanol, namely70% ethanol, had the best elution ability. So 70% ethanol was selected.

3.11 Determination of Elution Speed

The columns were filled with 50 g of pre-treated D₁₀₁ resin and afterthe dynamic absorption the column was eluted with 70% ethanol at speedsof 1, 2, and 3 BV/h, respectively. The content of the anthraquinones inthe ethanol solution was determined. The results are given in table 14.

TABLE 14 Table 14. Effect of different elution speed on the elutionability of anthraquinones Speed 1 BV/h 2 BV/h 3 BV/h Anthraquinones (mg)2180 2120 1960

From the table above, it was determined that the slower the elutionspeed, the better the desorption effect. The difference between 1 BV/hand 2 BV/h was not significant and 2 BV/h was selected in considerationof reducing processing time and increasing efficiency in large scaleproduction.

3.12 Desorption Curve

Based on above selected conditions, apply the sample solution onto acolumn filled with 50 g of pre-treated D₁₀₁ resin. Elute with ethanol.Collect the ethanol solution with every 10 ml as one sample. After15^(th) sample, collect 20 ml as one sample for further 20 samples.Determine the content of aurantio-obtusin by HPLC and prepare theelution curve. Results are given in table 15.

TABLE 15 Table 15. Desorption curve of aurantio-obtusin in Cassiaextracts on D₁₀₁ macroporous resin Sample 1 2 3 4 5 6 7 Aruantio- 0.7627 250 281 60 40 26 obtusin (mg) Sample 8 9 10 11 12 13 14 Aruantio-2.84 1.84 1.2 0.76 0.48 0.0372 0.0276 obtusin (mg) Sample 15 16 17 18 1920 Aruantio- 0.0108 0.0081 0.0062 0.0052 0.0030 0.0040 obtusin (mg)

From the table the 18^(th) sample showed a very low content of theanthraquinones, so the 18^(th) sample (200 ml) was decided as theelution end point, i.e. 4 BV of the eluent to be used.

3.13 pH1 Influence on the Yield of Anthracuinones

The test solution was adjusted to pH 4, 5, and 6 (pH 5 in the originalsolution), respectively and the effect of pH on anthraquinone contentdetermined—see table 16.

TABLE 16 Table 16. pH influence on the yield of anthraquinones pH value4 5 6 Total 2240 2220 1740 chemicals (mg)

From the table above, the acidic pH of around 4/5 appeared moreeffective than a pH of around 6.

3.14 The Effect of the Diameter to Height Ratio of the Resin Bed on theYield of the Anthraquinones

Based on the above selected conditions for absorption and elution, 3columns (20 mm×300 mm) were filled with the pre-treated macroporousresin 17 ml, 34 ml and 50 ml to have a diameter to height ratio 1:5,1:10, and 1:20, respectively. 1.4 BV of the sample solution was appliedto the column and eluted as mentioned. The results are given in table17.

TABLE 17 Table 17. The effect of the diameter to height ratio of theresin bed on the yield of the anthraquinones Diameter to height ratio ofresin bed 1:5 1:10 1:20 Group compounds 1773 1424 2081 (mg)

The result showed that when the diameter to height ratio of resin bedwas increased above 1:10 to 1:20, the content of the anthraquinonesincreased.

3.15 Further Purification

There are water soluble gum materials in the Cassia seeds. It was foundthese gums could be dissolved in water and low concentrations of ethanolwithout eluting the anthraquinones. They could be effectively eluted outby 20% ethanol while anthraquinones could not be eluted out at thisconcentration. Accordingly a wash step with water followed by elutionwith 20% ethanol to get rid of the gums was introduced.

In addition, re-dissolving the extract in 95% of ethanol and filteringremoved water soluble residues.

3.16 Pilot Scale Production of the Extracts

Based on a process employing the parameters selected above, threebatches of pilot scale extractions were conducted in order to confirmthe feasibility of the process in the large scale production. The resultare given in table 18

TABLE 18 Table 18. Pilot scale production of Cassia extracts Batch NoAnthraquinones % Aurantio-obtusin % Extract yield % 030815 45.5 4.5 0.97030818 51.0 5.4 1.07 030821 53.4 5.8 0.98 Average 50.0 5.2 1.01

A plant extract obtainable by the method described above could becharacterized as having an anthraquinone content in excess of 45% and anaurantio obtusin content in excess of 4.5%, measured as and calculatedon the basis of the total aglycone after acid hydrolysis. It wassubjected to further quality analysis as set out in 4.0 below:

4.0 Quality Specifications Product Code: PYN 22

This product extracted from the seeds of Cassia obtusifolia usingethanol as per the methodology described above was determined to containa minimum of 4.5% aurantio-obtusin (total of the aglycone in both freeform and in combined form which could be made to be free form withhydrolysis), and have a total anthraquinone content of at least 50%. Theamount of obtusifolin was at least 0.25%, more typically at least 0.5%.Based on pilot scale averages the anthraquinone content is about 50%,the aurantio-obtusin content is about 5.2% and the obtusifolin content(based on Table 19) is about 0.6% (all figures plus/minus 10%).

It can be characterized by way of HPLC as described under 3.1.1-3.1.4.

Weigh 10 mg sample, add 20 ml of methanol and 10 ml of 5% HCl, andreflex for 30 minutes. Extract the solution with 30 ml ether and removethe ether from the solution. Dissolve the residue in methanol in a 25 mlvolumetric flask, shake well and add further methanol to volume. Filterthrough a 0.22 μm micro membrane before injection.

Inject 20 μl of sample solution, determine peak area of each component,substitute the value in the linear equations to calculate the content,the result is as follows (unit: mg/g):

TABLE 19 Series No. Aurantion-obtusin Obtusifolin 1 57.599 6.785 245.178 5.231 3 52.433 6.189 Average 51.737 6.068

The HPLC profile is given in FIG. 4.

It can also be viewed by TLC.

Methodology:

Take 10 mg of the 4 batches of the samples, respectively, add methanol10 ml, under ultrasonic vibration for 60 mins, filter and the filtrateas the sample solution;

Dissolve appropriate amount of Aurantion-obtusin and Obtusifolin inmethanol as the reference solution.

Perform TLC based on the methods described in the Chinese Pharmacopoeia(Appendix VI B). Apply 1 μl of each of the above solutions onto a plate.(Activation at 105° C. for 30 mins before use).

Developer:

Petroleum ether (30° C.-60° C.): n-hexane:ethyl formate:formate(1:3:1.5:0.01).

Pre-saturate TLC tank for 15 mins before the plate is put in.

Developed TLC plates.

Examine the TLC plates at UV365 nm and 254 nm. The results areillustrated in FIGS. 5 and 6 respectively.

It can be further characterized as follows:

1. Appearance Brown powder with slightly unique smell. 2. Content ofAurantio-obtusin >5% (HPLC) measured as and calculated on the basis ofthe total aglycone after acid hydrolysis 3. Content of obtusifolin >0.5%(HPLC) measured as and calculated on the basis of the total aglyconeafter acid hydrolysis 4. Loss on Drying <9.0% (1 g sample, 105° C., 2 h)5. Purity Test (1) Heavy Metals <10 ppm (2) Arsenic <1 ppm 6. Microbialcounts <3 × 1³ cfu/g 7. Moulds and Yeasts <1 × 10³ cfu/g 8. ColiformsNegative 9. Composition 100% Cassia obtusifolia extract.

The dried extract can be used to fill capsules, in a unit dosage form,containing e.g. 250 mg of extract such that a daily does of 500 mg canbe easily given. This is equivalent to a 50 g of the raw material.

In animal studies the extract has been demonstrated to exhibit ananti-obesity activity.

5.0 Efficacy Studies Anti-Obesity Effect Materials and Methods Sample:

The sample PYN22 was prepared using the protocol described above. It isa dark brown powder.

Experimental Animals and Feeding Materials:

Seventy five male SD rats, weighting 140 g±10 g each, were provided bythe Experimental Animal Centre, Sichuan University. The animal lived inan environment with a temperature of 22-24° C. and a humidity of 65-70%.They were fed on two diets:

-   -   i) Basic Feeding Materials: Barley powder 20%, dehydrolized        vegetables 10%, bean powder 20%, yeast 1%, bone powder 5%, corn        flour 15%, wheat bran 16%, fish powder 10% and salt 2%.    -   ii) High fat and high nutrition feeding materials: To 100 g of        the basic feeding materials were added 10 g milk powder, 10 g        lard, one egg, 10 drops of concentrated fish liver oil and 50 g        of fresh bean sprouts.

Dose Selection:

Based on a recommended daily dose of 0.5-0.6 g for humans, three doses,0.05 g/kg·bw/day, 0.15 g/kg·bw/day and 0.30 g/kg·bw/day, were selectedfor the animal study, which were equivalent to 5 fold, 15 fold and 30fold of the recommended human daily dose, respectively.

Equipments and Solvents:

Electronic balance (BL610)

Experimental Methods:

The rats were randomly divided into five groups:

1. A normal control group

2. A high fat control group; and

3. Three testing groups.

Each was given the test sample at the dose of 0.05 g/kg·bw/day, 0.15g/kg·bw/day and 0.30 g/kg·bw/day, respectively.

The Normal control group was fed with the basic feeding materials. Allother groups were fed with high fat and high nutrition materials. Allthe animals were free to take the food and drink. The test samples weregiven once a day, via the intragastric route, to the three testinggroups at the dose mentioned above (the volume of the liquid was 1% ofthe body weight of the rat).

The normal control group and high fat control group were given distilledwater instead of the testing sample. The experiment lasted for 36 days.The animals were then dissected.

Observation Criteria:

Body weight;

Body fat weight (testis and kidney surrounding fat) and Body fat/bodyweight ratio were determined and side effects noted.

Data Analysis:

Statistical software was used for variance analysis.

Results

The effect on body weight is illustrated in Table 20 below.

TABLE 20 Table 20. The effect of Cassia extract (PYN-22) on the bodyweight in rats Body weight (g) After Weight increase Before exp. Middleterm exp. (g) Normal control 15 141.2 ± 9.1 222.7 ± 19.3 287.7 ± 25.3146.3 ± 29.6 High fat control 15 141.4 ± 10.9 239.7 ± 22.5* 326.5 ±37.5** 184.9 ± 39.1** 0.05 g/kg.bw/d 15 140.2 ± 10.1 223.2 ± 21.7^(♦)293.1 ± 31.1^(♦) 152.9 ± 29.1^(♦) 0.15 g/kg.bw/d 15 140.7 ± 10.6 219.4 ±32.8^(♦) 290.2 ± 39.2^(♦) 150.1 ± 36.3^(♦) 0.30 g/kg.bw/d 15 139.9 ± 9.9248.9 ± 23.5 318.9 ± 51.3 179.3 ± 53.7 Compared with normal controlgroup: **P < 0.01; *P < 0.05. Compared with high fat control group:^(♦♦)P < 0.01; ^(♦)P < 0.05.

From table 20, the results showed that in the middle term of theexperiment, the average body weight of the rats in the high fat controlgroup was significantly higher than that in the normal control group,which meant that the fat animal model was set successfully.

Three groups took the testing materials for 36 days and in two of them,at the dose of 0.05 g/kg·bw/day and 0.15 g/kg·bw/day, their average bodyweight and increased body weight were significantly lower than that inthe high fat control groups, which meant that the testing sample couldreduce the animal body weight. No diarrhea and hair loss were observedduring the experiment.

The Physical Measurement on the Rat

The effect on body fat weight is illustrated in Table 21 below.

TABLE 21 Table 21. The effect of Cassia extract (PYN-22) on the body fatweight in rats Final body Body fat wet Body fat/body Groups Animal No.weight (g) weight (g) weight ratio Normal control 15 287.7 ± 25.3 6.1 ±1.7 0.022 ± 0.005 High fat control 15 326.5 ± 37.5** 8.7 ± 2.1** 0.027 ±0.006* 0.05 g/kg.bw/d 15 293.1 ± 31.1^(♦) 6.5 ± 1.5^(♦♦) 0.022 ±0.006^(♦) 0.15 g/kg.bw/d 15 290.2 ± 39.2^(♦) 6.1 ± 1.9^(♦♦) 0.021 ±0.005^(♦♦) 0.30 g/kg.bw/d 15 318.9 ± 51.3 7.2 ± 2.6 0.022 ± 0.006♦Compared with the normal control group: **P < 0.01. Compared with thehigh fat group: ^(♦♦)P < 0.01; ^(♦)P < 0.05.

From the table 21 above it can be seen that, at the completion of theexperiment, both the body fat content, and body fat/body weight ratio ofthe high fat control group were significantly higher than that in thenormal group (P<0.01). This demonstrates that the fat animal model wasset successfully.

The body fat content in two testing groups (0.05 g/kg·bw/day and 0.15g/kg·bw/day) was significantly lower than that in the high fat controlgroup (P<0.01);

The body fat/body weight ratio in all three testing groups wassignificantly lower than that in the high fat control group (P<0.05).This demonstrated that the test sample could reduce the body fat contentof the animals, i.e. it may act as an anti-obesity agent.

Conclusion

The body weight, body fat content, body fat/body weight ration in highfat control group were significantly higher than that in the normalcontrol group.

The body weight, body fat content in two testing groups (0.05g/kg·bw/day, 0.15 g/kg·bw/day) was significantly lower than that in thehigh fat control group (P<0.01).

The body fat/body weight ratio in all three testing groups wassignificantly lower than that in the high fat control group (P<0.05).

All results suggested that the test sample had an anti-obesity effect inrat.

6.0 Toxicity Study 6.1 Acute Toxicity Study

The maximum dose of intra-gastric administration of the extract ofCassia-seeds was 16.50 g/kg in a single dose. The LD₅₀ was 20.84 g/kgwhen administrated twice (interval 4 hours) and it was equivalent to2511 times of proposed clinical dose (kg/bw). So it is safe for adult totake 0.5 g Cassia extract (PYN-22) in two capsules, equivalent to 50 graw material, per day.

6.2 Long Term Toxicity Study

The rats were intragastriclly given the extract of Cassia seed and therats were dissected in the 16^(th), 26^(th) week of the medication, andthe 4^(th) week after the medication, respectively. There were noobvious histopathological changes in the organs from the testing groupanimals compared with those from the animals in the normal controlgroup.

7.0 Further Experimental Analysis to Consider a) Body Weight and BodyFat; b) Metabolic Parameters; and c) Liver Indicators

Experimental design: Forty C57 black ob/ob mice were allocated to fivegroups of eight for study over 40 days as follows:

-   -   Lean diet—no treatment;    -   High fat diet—no treatment;    -   High fat diet—100 mg/kg/OD PYN22;    -   High fat diet—300 mg/kg/OD PYN22;    -   High fat diet—3 mg/kg/OD Rosiglitazone (positive control).

Mice were weighed twice per week and food intake measured dailythroughout the study. Any laxative effects of treatments were visuallyexamined and recorded.

Oral glucose tolerance tests were conducted on Day 7, Day 21 and Day 35.

Energy expenditure was measured at Day 14.

Fasting plasma insulin and lipids were measured on Days 7, 21 and 35.

Three days before sacrifice, 100 ul blood samples were taken from allanimals to provide plasma glucose, insulin, free fatty acid,triglycerides, cholesterol and HDL-cholesterol.

At sacrifice, body mass and fat mass were calculated; the sameparameters were also calculated for liver, heart and white adiposetissues.

Animal well-being/safety measures: All animals in each of the studygroup survived the treatment regimens until the scheduled sacrificedate. Each group of animals was examined on a daily basis and nolaxative effect was recorded in any treatment group.

Data Analysis: Body Weight & Body Fat:

Over the course of the study, animals in the Rosiglitazone control grouphad an increased body weight that exceeded the other four groups (FIG.7A), as reflected clearly in weight gain in the study period (FIG. 7B).However, the body fat gained was not significantly different than thetreatment free high fat group. Only high levels of PYN22 resulted insignificantly less body fat gain relative to the high fat control, and300 mg OD PYN22 seemed to prevent body fat increasing above lean controllevels, despite high fat intake in this group (FIG. 7C).

In terms of circulating lipids, there was a possible dose-response trendof cholesterol reduction by PYN22 but only Rosiglitazone gave astatistically significant reduction on total cholesterol in high fatdiet groups (FIG. 8A). However, 100 mg OD PYN22 gave a significantreduction in HDL cholesterol that became highly significant at 300 mg ODand surpassed the effects of Rosiglitazone (FIG. 5B).

Metabolic Parameters:

In terms of insulin levels, PYN22 consistently lowered fasting insulinin animals on a high fat diet, bringing levels part-way towards thosereceiving a lean diet, but not achieving the effects of Rosiglitazone, amarketed anti-diabetic therapy. (FIGS. 9A-9C).

In oral glucose tolerance tests (OGTTs), PYN22 reversed the effects ofthe high fat diet towards the lean diet group (FIGS. 10A, 10B and 10C)in a dose-dependent trend at all time points in the study but withoutstatistical significance, and with less effect than Rosiglirazone.

Liver Indicators:

PYN22 had no effect on ALT levels (but reduced AST levels in adose-dependent trend but without statistical significance—FIGS. 11A and11B). PYN22 also reduced actual and relative (% body weight) liverweight,—(FIGS. 11C and 11D) whereas Rosiglitazone had no effect.

PYN22 had little effect on liver glycogen levels (FIG. 11E) but reducedliver triglycerides substantially (FIG. 11F), although notsignificantly.

The above results provide credible evidence that in addition to treatingobesity the extract of the invention may also be used to treat metabolicdisease or liver disease.

SUMMARY

The effect on lipid levels and on metabolic indicators is manifest in astatistically significant reduction in body fat.

The data is additional suggestive of further clinical application in thetreatment of:

-   -   Chronic inflammation of the liver resulting from metabolic        overload and fat deposition;    -   Fatty liver; and    -   Fibrogenesis.

1. A selective alcoholic plant extract, from a Cassia spp, comprising byanalysis: i) at least 2% aurantio-obtusin as determined by hplcanalysis; ii) at least 0.2% obtusifolin as determined by hplc analysis;and iii) at least 25% total anthraquinone glycoside as determined byspectrophotometry; and wherein iv) the ratio of aurantio-obtusin toobtusifolin is between 5:1 and 30:1 v) the extract has had lipo-solublegums and water soluble gums removed and has been selectively purified,by at least a factor of 50, as compared to a starting plant material. 2.The plant extract as claimed in claim 1 wherein the Cassia spp. isselected from Cassia obtusifolia or Cassia tora.
 3. The plant extract asclaimed in claim 1 wherein the extract is obtained from Cassia seed. 4.The plant extract as claimed in claim 1 further comprising at least 5%aurantio-obtusin by hplc analysis.
 5. The plant extract as claimed inclaim 4 further comprising at least 10% aurantio-obtusin by hplcanalysis.
 6. The plant extract as claimed in claim 1 comprising islimited to about 0.5% obtusifolin by hplc analysis.
 7. The plant extractas claimed in claim 1 having ratio of aurantio-obtusin to obtusifolin ofbetween 8:1 and 16:1.
 8. The plant extract as claimed in claim 1 whereinthe total anthraquinone comprises chrysophanol, physcion, obtusifolin,emodin, and aruantio-obtusin.
 9. The plant extract as claimed in claim 1further comprising at least 45% total anthraquinone byspectrophotometry.
 10. The plant extract as claimed in claim 1 which hasbeen purified by at least a factor of 75 as compared to a starting plantmaterial.
 11. The plant extract as claimed in claim 1 which is anethanolic extract.
 12. The plant extract as claimed in claim 11 which isa refluxed extract.
 13. The Botanical Product comprising a plant extractas claimed in claim
 1. 14. The Botanical Product as claimed in claim 13which is a Botanical Drug Substance or a Botanical Drug.
 15. The BotanicProduct as claimed in claim 14 which is provided in unit dosage form.16. The Botanical Product as claimed in claim 15 wherein the unit dosageform is a capsule for oral delivery.
 17. The Botanical Product asclaimed in claim 15 which comprises a unit dose of from 50-5000 mg ofextract equivalent to 5 to 500 g of raw material.
 18. The BotanicalProduct as claimed in claim 17 in the form of a dietary supplement, foodstuff, or cosmetic.
 19. A method for obtaining a pharmaceutically activeextract from a Cassia spp as claimed in claim 1 comprising: i) Selectingthe Spp and plant material; ii) Comminuting the plant material; iii)Subjecting the comminuted plant material to an alcoholic extraction; iv)Conducting a separation step to remove lipo-soluble and water solublegums; and v) Conducting a purification step.
 20. The method as claimedin claim 19 wherein the plant spp is Cassia obtusifolia.
 21. The methodas claimed in claim 19 wherein the alcoholic extraction is an ethanolicextraction.
 22. The method as claimed in claim 21 wherein the ethanolicextraction comprises a reflux extraction.
 23. The method as claimed inclaim 21 wherein the ethanol has strength of from 50-80%.
 24. The methodas claimed in claim 23 wherein the reflux extraction comprises a repeatextraction process.
 25. The method as claimed in claim 19 comprising acentrifugation step or a partitioning step with a lipo-soluble solvent.26. The method as claimed in claim 19 wherein a recovered extract isconcentration adjusted with distilled water to a ratio of greater than1:1, material to solution.
 27. The method as claimed in claim 19comprising a resin absorption separation and purification process. 28.The method as claimed in claim 27 which utilizes a macroporus resincolumn.
 29. The method as claimed in claim 28 wherein the macroporusresin is non-polar resin.
 30. The method as claimed in claim 29 whereinthe macroporus resin is D₁₀₁.
 31. The method as claimed in claim 28wherein the column has a diameter to height ratio in excess of 1:2. 32.The method as claimed in claim 27 wherein for absorption, the eluate isapplied to the column in an amount of less than 4 BV and at a rate ofless than 2 BV/hr.
 33. The method as claimed in claim 27 whereindesorption is conducted with up to 10 BV of from 60-80% ethanol at aspeed of from 1-5 BV/hr.
 34. The method as claimed in claim 27 whereinbetween absorption and desorption the column is washed to remove solublegums.
 35. The method as claimed in claim 34 wherein the wash isconducted using water followed by low concentration ethanol.
 36. Themethod as claimed in claimed in claim 35 wherein the wash step is withfrom 1-8 BV water followed by from 1-8 BV of 10-30% ethanol at a speedof from 1-5 BV/hr.
 37. The method as claimed in claim 19 furthercomprising drying and powdering the extract.
 38. The use of selectivealcoholic extract of a Cassia spp as claimed in claim 1 in themanufacture of a medicament for the treatment of obesity, metabolicdisease, or liver disease.
 39. The use of selective alcoholic extract ofa Cassia spp as claimed in claim 1 in the manufacture of a medicamentfor the treatment of Chronic inflammation of the liver resulting frommetabolic overload and fat deposition, fatty liver, or fibrogenesis. 40.The use of selective alcoholic extract of a Cassia spp as claimed inclaim 1 in the manufacture of a cosmetic preparation for the treatmentof obesity.
 41. The use of selective alcoholic extract of a Cassia sppas claimed in claim 1 in the manufacture of a food stuff or dietarysupplement.
 42. A method for the treatment of obesity, metabolicdisease, or liver disease comprising administering a plant extract asclaimed in claim
 1. 43. The method as claimed in claim 42 wherein themetabolic disease is diabetes.
 44. The method as claimed in claim 42wherein the liver disease is fatty liver.
 45. The method for thecosmetic treatment of obesity comprising administering a plant extractas claimed in claim
 1. 46. A method for supporting weight losscomprising administering a food stuff or dietary supplement comprising aplant extract as claimed in claim
 1. 47. A plant extract characterizedin that it has an HPLC fingerprint substantially as shown in FIG. 4 withpeaks corresponding to aurantion-obtusin and obtusifolin.
 48. A plantextract characterized in that it has an TLC fingerprint substantially asshown in FIG. 5, 365 nm, or FIG. 6, 254 nm, with spots corresponding toaurantion-obtusin and obtusifolin.